Deciphering cytokine-driven ADP-ribosylation signaling networks via Af1521-based mass spectrometry analysis of labile Glu/Asp-linkages - PubMed
an hour ago
- #mass spectrometry
- #innate immunity
- #ADP-ribosylation
- ADP-ribosylation (ADPr) targets nine amino acid residues, but labile glutamate/aspartate-linked ADPr (Glu/Asp-ADPr) is challenging to detect with conventional mass spectrometry (MS).
- Ester-linked Glu/Asp-ADPr is lost under alkaline conditions, high temperatures, and hydrolysis by wildtype Af1521.
- An acidic enrichment workflow using an Af1521 mutant preserves Glu/Asp-ADPr, enabling site-specific, system-wide MS analysis.
- In cytokine-stimulated A549 and HeLa cells, over 600 Glu/Asp- and over 200 Cys-ADPr sites were identified.
- Glu/Asp-ADPr marks cytoplasmic, immune-related protein networks, while Ser-ADPr is nuclear.
- Quantitative profiling showed reproducible, cell type- and treatment-specific ADPr patterns.
- PARP10-mediated Glu/Asp ADPr of ubiquitin suggests direct crosstalk with ubiquitin signaling pathways.
- Interferon treatments revealed conserved antiviral PARP networks extensively modified on Glu/Asp residues.
- The study establishes a robust MS workflow and provides a resource of site-specific ADPr events in innate immune signaling.